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miR-503 suppresses tumor cell proliferation and metastasis by directly targeting RNF31 in prostate cancer
Biochemical and Biophysical Research Communications xxx (2015) 1e7
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miR-503 suppresses tumor cell proliferation and metastasis by directly targeting RNF31 in prostate cancer Jia Guo, Xiuheng Liu*, Min Wang Department of Urology, Renmin Hospital of Wuhan University, Wuhan University, Jiefang Road 238, Wuhan 430060, Hubei, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 July 2015 Accepted 25 July 2015 Available online xxx
Microarray data analyses were performed to search for metastasis-associated oncogenes in prostate cancer (PCa). RNF31 mRNA expressions in tumor tissues and benign prostate tissues were evaluated. The RNF31 protein expression levels were also analyzed by western blot and immunohistochemistry. Luciferase reporter assays were used to identify miRNAs that can regulate RNF31. The effect of RNF31 on PCa progression was studied in vitro and in vivo. We found that RNF31 was significantly increased in PCa and its expression level was highly correlated with seminal vesicle invasion, clinical stage, prostate specific antigen (PSA) level, Gleason score, and BCR. Silence of RNF31 suppressed PCa cell proliferation and metastasis in vitro and in vivo. miR-503 can directly regulate RNF31. Enforced expression of miR-503 inhibited the expression of RNF31 significantly and the restoration of RNF31 expression reversed the inhibitory effects of miR-503 on PCa cell proliferation and metastasis. These findings collectively indicated an oncogene role of RNF31 in PCa progression which can be regulated by miR-503, suggesting that RNF31 could serve as a potential prognostic biomarker and therapeutic target for PCa. © 2015 Published by Elsevier Inc.
Keywords: miR-503 RNF31 Metastasis Prostate cancer Proliferation Overall survival
1. Introduction Prostate cancer (PCa) is the most frequently diagnosed malignancy and the second leading cause of malignancy-related deaths among men in developed countries [1]. Despite the unceasing biomedical research efforts, PCa continues to pose a major public health problem [2]. It is well known that prostate specific antigen (PSA) still remains, in spite of the ongoing criticism, one of the most extensively applied PCa molecular markers [3]. Although considerable advances in diagnosis and adjuvant therapy of PCa have been made, many patients with PCa will develop metastases, the overall survival rate has not been improved markedly [4e6]. Although several clinical factors, such as PSA and Gleason score, may provide some prognostic utility in the treatment settings, there are currently no definitive clinical methods that can reliably predict the responses to clinical therapies for patients with PCa [7e9]. Therefore, there is an urgent need for prognostic biomarkers to strengthen the efficiency of early diagnosis and to improve the therapeutic strategies of PCa. RNF31 is a RING finger protein, also known as HOIL-1 interacting
* Corresponding author. E-mail address: [email protected] (X. Liu).
protein [10,11]. The interaction of RNF31 and RBCK1 occurs through binding of the ubiquitin-associated domain of RNF31 with the ubiquitin-like domain of RBCK1 [12]. This complex has the unique ability to catalyze the formation of linear peptide bonds between the amino-terminal methionine of one ubiquitin molecule and the carboxyl-terminal glycine of the adjacent ubiquitin [13]. RNF31 could constitute a valuable diagnostic tool and/or a drug target for ERa-positive breast cancers [14,15]. Inhibition of RNF31 activity can affect p53 signaling to enhance the effect of chemotherapy [14,15]. It is well known that miRNAs are small non-coding RNA molecules consisting of 19e23 nucleotides that can regulate various biological processes. More than 60% of protein-coding genes may be targeted by miRNAs mainly through blocking mRNA translation or degrading mRNAs by binding to the 30 untranslated regions (UTR) of specific mRNAs [16]. An expanding body of evidence supports a role for miRNAs in disease initiation, promotion, and progression [17e20]. In view of the extensive molecular functions of miRNAs and RNF31, it will be necessary to investigate the molecular mechanism and role of RNF31 in PCa progression and to find whether or not RNF31 expression can be regulated by a specific miRNA in PCa. Here we have shown that RNF31 expression is significantly increased in primary PCa tissues and PCa metastatic tissues. We have shown that specific knockdown of RNF31using RNA
http://dx.doi.org/10.1016/j.bbrc.2015.07.127 0006-291X/© 2015 Published by Elsevier Inc.
Please cite this article in press as: J. Guo, et al., miR-503 suppresses tumor cell proliferation and metastasis by directly targeting RNF31 in prostate cancer, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.127
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interference resulted in decreased PCa cell invasion in vitro and also metastasis in vivo. Our data demonstrate for the first time that RNF31 is overexpressed in aggressive PCa and that this expression correlates with PCa progression. Our results also revealed a correlation between high RNF31 levels and reduced miR-503 expression in the PCa tissues. We further identified that the miR-503 can regulate the invasiveness of PCa cells and the metastasis of PCa xenografts by regulating RNF31. Collectively, these results provided potential effective molecular biomarkers for the prognosis, and developed effective potential therapeutic targets for the treatment of PCa. 2. Materials and methods 2.1. Clinical specimens 140 PCa and paired adjacent normal tissues were obtained from the Renmin hospital of Wuhan university, which underwent radical prostatectomy between 2002 and 2009. None of the patients had received androgen deprivation treatment, chemotherapy or radiation therapy prior to the surgery. All patients' informed consents were obtained. This study was approved by the research ethics committee of our hospital. 2.2. Cell lines PCa cell lines PC-3 and LNCaP were grown in RPMI 1640 (Life Technologies, CA) with 0.023 IU/ml insulin and 10% FBS (Invitrogen) in 5% CO2 cell culture incubator. 2.3. Plasmids and cell transfection A cDNA sequence containing one pre-miR-503 unit was inserted into pcDNA3.1 (Promega, Madison, WI, USA). The RNF31 shRNA was designed with a shRNA designer tool. Two strands were annealed, followed by insertion into pcDNA6.2-GW/EmGFP-miR vector. The RNF31 cDNA containing the coding sequence was cloned by PCR, and the PCR product was cloned into the pcDNA3.1 vector. The insert was confirmed by DNA sequencing. 2.4. Colony formation assay In colony formation assay, the cells were seeded on 35-mm dishes. The cells were fixed in methanol, and then stained with crystal. Finally, positive colony formation (>50 cells/colony) was counted. 2.5. Cell migration and invasion assay Transwell migration and invasion assays were performed with 8.0-mm pore according to the manufacturers' instructions (BD Bioscience, CA). The PCa cell migration and invasion assays were performed with uncoated (migration) and coated Matrigel (invasion). The migrated and invaded PCa cells in the membrane were fixed and stained, and the cells were counted under a microscope.
2.7. RNA extraction and qRT-PCR analyses Total RNA was extracted using Trizol Reagent according to the manufacturer's protocol. The expression level of miR-503 was measured by TaqMan miRNA assays (Applied Biosystems, CA, USA) according to the provided protocol, miRNA U6 was used for normalization. RNF31 expression was measured by SYBR green qPCR assay and b-actin was used as an endogenous control. 2.8. Western blot analysis Protein concentration was measured by use of the BCA reagent kit (Merck). The protein was resolved by SDS-PAGE and transferred to a PVDF membrane, which was probed with specific primary antibody against RNF31. b-actin was blotted to show equal protein loading. 2.9. Immunohistochemistry After deparaffinization and rehydration, the tissues were washed by phosphate-buffered saline and treated with 3% H2O2 in methanol for 10 min. After being washed with distilled water, the tissues were subjected to antigen retrieval in citrate buffer and stained overnight with rabbit polyclonal anti-RNF31 antibody. The sections were incubated with goat anti-rabbit IgG for 30 min and developed with diaminobenzidine. RNF31 protein level was classified semiquantitatively combining the proportion and intensity of positively stained immunoreactive cells [21,22]. The sum of the staining intensity score and the percentage score was used to define the RNF31 protein expression levels: 0e2, low expression and 3e4, high expression [21,22]. 2.10. Prostate tumor xenograft studies We established xenografts in nude mice with the stable expressing miR-503 cells, RNF31 knockdown cells, and control cells. PCa cells were implanted into the dorsal flank of male Athymic nude mice subcutaneously. Tumor size was measured biweekly, and tumor volumes were calculated using the formula: Volume (mm3) ¼ [width2 (mm2) length (mm)]/2. Mice with tumors were killed 7 weeks after the inoculation. The xenograft tumors, and the cervical lymph nodes were collected and tumor weights were measured. DNA extraction of the cervical lymph nodes and human alu sequence PCR amplification were performed as described previously [23]. 2.11. Statistical analysis For continuous variables, Student's t-test was performed. Spearman correlation test was chosen for examining the correlations between RNF31 expression level and the clinical and pathological variables. Survival curves were carried out by the KaplaneMeier method and evaluated using the log-rank test. Identified factors were associated with survival by the Cox proportional hazard regression model. P < 0.05 was considered statistically significant. Statistical analysis was performed using SPSS 17.0 software.
2.6. Luciferase reporter assay 3. Results In brief, the miR-503-binding site in the RNF31 30 -UTR region (wild or mutant-type) was cloned downstream of the firefly luciferase gene in a pGL3-promoter vector. The luciferase assay was performed following the manufacturer's protocol. Luciferase activity was measured using the dual luciferase reporter assay system (Promega, Madison, WI).
3.1. RNF31 is a potential metastasis associated gene and is associated with PCa progression To identify whether any significant difference of RNF31 expression level exists in metastatic PCa, primary PCa, and normal
Please cite this article in press as: J. Guo, et al., miR-503 suppresses tumor cell proliferation and metastasis by directly targeting RNF31 in prostate cancer, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.127
J. Guo et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e7
prostate samples, the microarray data from some available datasets were analyzed [24e28]. The data showed that RNF31 mRNA was significantly increased in primary PCa compared with normal prostate samples (Fig. 1AeC). Similarly, elevated RNF31 mRNA was found in metastatic PCa relative to primary prostate tissues (Fig. 1DeF). We further found that RNF31 was significantly with higher PCa grade, lymph node metastasis, and higher stage (Fig. 1GeI). Collectively, RNF31 is a potential metastasis associated gene in PCa. RNF31 protein expression was high in 4 of 50 patients with benign prostate hyperplasia (BPH) and 92 of 140 patients with PCa. RNF31 protein expression was overexpressed in PCa tissues compared with the BPH tissues, and the difference was statistically significant. As shown in Fig. 1J, the RNF31 staining was localized within the cytoplasm of immunoreactive prostate cells. The immunohistochemical analysis showed weak or no reactivity in normal prostate samples but strong staining in the aggressive PCa tissues. We further studied the association between RNF31 protein expression status and clinicopathological factors in PCa. The associations between RNF31 protein expression and clinicopathological factors are shown in Table S1. High expression of RNF31 protein was significantly associated with seminal vesicle invasion, the higher
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level of preoperative PSA, the positive angiolymphatic invasion, BCR, and the higher Gleason score. We further found that the high RNF31 protein expression group had a shorter overall or BCR-free survival duration compared to the low RNF31 expression group. Univariate and multivariate analysis showed that RNF31 protein expression was an independent prognostic factors for BCR-free and overall survival of patients with PCa (Tables S2 and S3). Taken together, these results indicated RNF31 overexpression is significantly associated with PCa progression and poor prognosis. 3.2. Silence of RNF31 inhibits PCa cell colony formation, migration and invasion In order to study the functional significance of RNF31 upregulation in PCa, we reduced RNF31 expression level in PCa cells and investigated its impacts on cell colony formation, migration, and invasion. We employed stable knockdown strategy targeting RNF31 in LNCaP and PC-3 cell lines. The efficiency of silence of RNF31 was investigated by western blots. We found significant decrease in colony formation in RNF31 knockdown cells compared with control cells (Fig. 2A). We also found that silence of RNF31 dramatically inhibited PC-3 and LNCaP cell migration and invasion (Fig. 2B, C). RNF31 protein was significantly decreased in the RNF31
Fig. 1. RNF31 is upregulated in PCa tissues and is correlated with PCa progression. A, B, C, D, E and F, Box plots represent RNF31 mRNA level in normal prostate, primary PCa and metastatic PCa tissues. G, H, and I, RNF31 is significantly upregulated in patients with higher PCa stage, lymph node metastasis, and higher PCa grade compared with patients with lower PCa stage, negative lymph node metastasis, and lower PCa grade respectively. J, Immunohistochemical analysis of RNF31 in benign prostate epithelia, less aggressive PCa, and aggressive PCa.
Please cite this article in press as: J. Guo, et al., miR-503 suppresses tumor cell proliferation and metastasis by directly targeting RNF31 in prostate cancer, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.127
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Fig. 2. miR-503 inhibits PCa cell proliferation, migration and invasion by targeting RNF31 in vitro. A, RNF31 knockdown can mimic the suppression of colony formation induced by miR-503 in PC-3 and LNCaP cells. B, RNF31 knockdown can mimic the suppression of migration activity induced by miR-503 in PC-3 and LNCaP cells. C, RNF31 knockdown can mimic the suppression of invasion activity induced by miR-503 in PC-3 and LNCaP cells. D, The efficiency of RNF31 knockdown and ectopic expression of miR-503 was confirmed at protein level by western blot.
knockdown cells relative to the control cells (Fig. 2D). 3.3. RNF31 as a direct target of miR-503 We used miRNA target prediction algorithms provided by miRWalk. After integrating the results, we choose miR-503 for further investigations due to its tumor suppressor properties in several malignancies [29e33]. The binding site for miR-503 at 30 UTR of RNF31 was depicted (Fig. 3A). We construct firefly luciferase reporter constructs with the 30 UTR of RNF31 mRNA, and transfected them into PCa cells with miR-503. We found that co-transfection with miR-503 can significantly reduce the luciferase activity when the construct contained the 30 UTR of RNF31 (Fig. 3B). Mutation of the binding sites can reverse the inhibitory effects. Collectively, these results indicated that RNF31 was a direct target of miR-503. We further conducted qRT-PCR and western blot in PCa cells to investigate whether ectopic expression of miR-503 can change RNF31 expression levels. RNF31 mRNA expression were dramatically inhibited in miR-503 transfectants as compared with control cells (Fig. 3C). We also found that RNF31 mRNA expression was inversely associated with the expression level of miR-503 (Fig. 3D). 3.4. miR-503 can regulate cell proliferation, invasion and migration by targeting RNF31 Our team investigate whether miR-503 can change the effects of RNF31 on PCa. We found that RNF31 upregulation can significantly abrogate the inhibition of PCa cell colony formation induced by miR-503 (Fig. 2A). Similarly, RNF31 upregulation can reverse the inhibition of PCa cell migration and invasion induced by miR-503
(Fig. 2B, C). The efficiency of miR-503 overexpression was investigated by western blots (Fig. 2D). We found that silence of RNF31 can result in similar effects induced by miR-503 overexpression in PCa cells (Fig. 2). 3.5. RNF31 plays an important role in PCa growth and metastasis in vivo To study the role of RNF31 on PCa growth in vivo, a murine xenograft model using stable RNF31 knockdown PCa cells was used. Silence of RNF31 can lead to significantly decreased tumor volume and weight relative to control group (Fig. 4AeD). Silence of RNF31 tumor tissues indicated reduced staining for RNF31 (Fig. 4E). Silence of RNF31 significantly inhibited PC-3 cell's ability to metastasize relative to control cells (Fig. 4F). Ectopic of miR-503 can result in attenuated metastasis relative to the control group, and lead to similar results induced by silence of RNF31 (Fig. 4). Our team did not find any metastases in control or experimental LNCaP xenografts. Taken together, these data suggested that RNF31 may play an important role in PCa growth and metastasis in vivo. 4. Discussion In this study, we want to identify RNF31 protein expression pattern and its association with PCa carcinogenesis, progression, and prognosis. Results indicated a statistically significant increase of RNF31 expression in PCa and metastatic PCa, indicating RNF31 plays an important role in PCa progression. To clarify the role of RNF31 in PCa, we studied the association of RNF31 staining with clinicopathological characteristics in PCa. Results indicated that RNF31 positive staining was significantly associated with PCa
Please cite this article in press as: J. Guo, et al., miR-503 suppresses tumor cell proliferation and metastasis by directly targeting RNF31 in prostate cancer, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.127
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Fig. 3. RNF31 is a direct target of miR-503 in PCa tissues. A, Computational analysis indicating that miR-503 potentially targeted RNF31. B, Relative luciferase activities were studied in PCa cells. C, Decrease in RNF31 mRNA expression by miR-503 was investigated using qRT-PCR. D, RNF31 mRNA was inversely associated with miR-503 in 140 pairs of PCa tissues using linear regression models.
Fig. 4. Ectopic expression of miR-503 inhibits tumor growth and metastasis via targeting RNF31 in vivo. A and B, Ectopic expression of miR-503 in PCa cells significantly inhibits tumor growth in a mouse xenograft model. C and D, Tumor weights of corresponding mouse xenograft models. E, RNF31 expression analysis was conducted at protein level by western blot. F, miR-503 played an important role in PCa metastasis in vivo via targeting RNF31.
Please cite this article in press as: J. Guo, et al., miR-503 suppresses tumor cell proliferation and metastasis by directly targeting RNF31 in prostate cancer, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.127
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progression. This is the first direct evidence of the association between RNF31 and clinicopathological factors of PCa. Given the significant association found between RNF31 and PCa progression, we further studied its prognostic role. Results indicated that positive staining of RNF31 was independently associated with unfavorable outcome of patients with PCa. The prognostic value of RNF31 was statistically significant in not only univariate analysis but also multivariate analysis adjusted for variables, which suggested RNF31 expression is a good biomarker to predict prognosis of PCa. These results above were the first to address the prognostic role of RNF31 in PCa. Proliferation and metastasis are two most common causes for the cancer-related death [14,34]. In order to identify the functions of RNF31, the stable RNF31 silence/overexpression PCa cell lines were constructed and the effects of them on cell proliferation, migration and invasion were studied. These results indicated that silence of RNF31 significantly inhibited the proliferation of the PCa cell lines in vitro. We found that PCa cells became less aggressive and invasive after transfected with the RNF31 knockdown construct, indicating that the RNF31 functioned as a metastasis associated gene in PCa. We also found that RNF31 knockdown can significantly inhibit tumor growth and metastasis in vivo. More and more evidence indicated miRNAs play essential roles in some processes, including differentiation, angiogenesis, proliferation, migration, and invasion. We then search the potential miRNAs which can regulate RNF31. Among of these miRNAs, miR-503 attracted our attention due to its role in several cancers. We found that miR-503 level was correlated inversely with RNF31 mRNA level. We further studied whether miR-503 can inhibit RNF31 expression level. We treated PCa cell lines with miR-503 and investigated RNF31 level. The results indicated that miR-503 can significantly negatively regulate RNF31 expression in PCa tissues. These results further indicated that the miR-503 can directly target RNF31. As expectedly, silence of RNF31 can lead to similar results induced by miR-503 overexpression in PCa. Furthermore, ectopic overexpression of RNF31 can significantly reverse the inhibitory effects of miR-503. Collectively, these results showed that miR-503 can inhibit PCa cell proliferation, migration and invasion by directly inhibiting RNF31. In conclusion, to our knowledge, this is the first study to describe the function of RNF31 in carcinogenesis, progression and prognosis of PCa. Our study also showed for the first time that miR503 may play its metastasis suppressive role through attenuation of RNF31 activity. Our investigation indicated for the first time that RNF31 could be an independent prognostic predictor to identify the poor BCR-free survival and overall survival PCa patients. Taken together, these results above suggested that RNF31 plays an important role in PCa progression and could serve as a viable therapeutic target. Acknowledgments None.
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2015.07.127. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.07.127.
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
miR-503 suppresses tumor cell proliferation and metastasis by directly targeting RNF31 in prostate cancer Jia Guo, Xiuheng Liu*, Min Wang Department of Urology, Renmin Hospital of Wuhan University, Wuhan University, Jiefang Road 238, Wuhan 430060, Hubei, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 July 2015 Accepted 25 July 2015 Available online xxx
Microarray data analyses were performed to search for metastasis-associated oncogenes in prostate cancer (PCa). RNF31 mRNA expressions in tumor tissues and benign prostate tissues were evaluated. The RNF31 protein expression levels were also analyzed by western blot and immunohistochemistry. Luciferase reporter assays were used to identify miRNAs that can regulate RNF31. The effect of RNF31 on PCa progression was studied in vitro and in vivo. We found that RNF31 was significantly increased in PCa and its expression level was highly correlated with seminal vesicle invasion, clinical stage, prostate specific antigen (PSA) level, Gleason score, and BCR. Silence of RNF31 suppressed PCa cell proliferation and metastasis in vitro and in vivo. miR-503 can directly regulate RNF31. Enforced expression of miR-503 inhibited the expression of RNF31 significantly and the restoration of RNF31 expression reversed the inhibitory effects of miR-503 on PCa cell proliferation and metastasis. These findings collectively indicated an oncogene role of RNF31 in PCa progression which can be regulated by miR-503, suggesting that RNF31 could serve as a potential prognostic biomarker and therapeutic target for PCa. © 2015 Published by Elsevier Inc.
Keywords: miR-503 RNF31 Metastasis Prostate cancer Proliferation Overall survival
1. Introduction Prostate cancer (PCa) is the most frequently diagnosed malignancy and the second leading cause of malignancy-related deaths among men in developed countries [1]. Despite the unceasing biomedical research efforts, PCa continues to pose a major public health problem [2]. It is well known that prostate specific antigen (PSA) still remains, in spite of the ongoing criticism, one of the most extensively applied PCa molecular markers [3]. Although considerable advances in diagnosis and adjuvant therapy of PCa have been made, many patients with PCa will develop metastases, the overall survival rate has not been improved markedly [4e6]. Although several clinical factors, such as PSA and Gleason score, may provide some prognostic utility in the treatment settings, there are currently no definitive clinical methods that can reliably predict the responses to clinical therapies for patients with PCa [7e9]. Therefore, there is an urgent need for prognostic biomarkers to strengthen the efficiency of early diagnosis and to improve the therapeutic strategies of PCa. RNF31 is a RING finger protein, also known as HOIL-1 interacting
* Corresponding author. E-mail address: [email protected] (X. Liu).
protein [10,11]. The interaction of RNF31 and RBCK1 occurs through binding of the ubiquitin-associated domain of RNF31 with the ubiquitin-like domain of RBCK1 [12]. This complex has the unique ability to catalyze the formation of linear peptide bonds between the amino-terminal methionine of one ubiquitin molecule and the carboxyl-terminal glycine of the adjacent ubiquitin [13]. RNF31 could constitute a valuable diagnostic tool and/or a drug target for ERa-positive breast cancers [14,15]. Inhibition of RNF31 activity can affect p53 signaling to enhance the effect of chemotherapy [14,15]. It is well known that miRNAs are small non-coding RNA molecules consisting of 19e23 nucleotides that can regulate various biological processes. More than 60% of protein-coding genes may be targeted by miRNAs mainly through blocking mRNA translation or degrading mRNAs by binding to the 30 untranslated regions (UTR) of specific mRNAs [16]. An expanding body of evidence supports a role for miRNAs in disease initiation, promotion, and progression [17e20]. In view of the extensive molecular functions of miRNAs and RNF31, it will be necessary to investigate the molecular mechanism and role of RNF31 in PCa progression and to find whether or not RNF31 expression can be regulated by a specific miRNA in PCa. Here we have shown that RNF31 expression is significantly increased in primary PCa tissues and PCa metastatic tissues. We have shown that specific knockdown of RNF31using RNA
http://dx.doi.org/10.1016/j.bbrc.2015.07.127 0006-291X/© 2015 Published by Elsevier Inc.
Please cite this article in press as: J. Guo, et al., miR-503 suppresses tumor cell proliferation and metastasis by directly targeting RNF31 in prostate cancer, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.127
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interference resulted in decreased PCa cell invasion in vitro and also metastasis in vivo. Our data demonstrate for the first time that RNF31 is overexpressed in aggressive PCa and that this expression correlates with PCa progression. Our results also revealed a correlation between high RNF31 levels and reduced miR-503 expression in the PCa tissues. We further identified that the miR-503 can regulate the invasiveness of PCa cells and the metastasis of PCa xenografts by regulating RNF31. Collectively, these results provided potential effective molecular biomarkers for the prognosis, and developed effective potential therapeutic targets for the treatment of PCa. 2. Materials and methods 2.1. Clinical specimens 140 PCa and paired adjacent normal tissues were obtained from the Renmin hospital of Wuhan university, which underwent radical prostatectomy between 2002 and 2009. None of the patients had received androgen deprivation treatment, chemotherapy or radiation therapy prior to the surgery. All patients' informed consents were obtained. This study was approved by the research ethics committee of our hospital. 2.2. Cell lines PCa cell lines PC-3 and LNCaP were grown in RPMI 1640 (Life Technologies, CA) with 0.023 IU/ml insulin and 10% FBS (Invitrogen) in 5% CO2 cell culture incubator. 2.3. Plasmids and cell transfection A cDNA sequence containing one pre-miR-503 unit was inserted into pcDNA3.1 (Promega, Madison, WI, USA). The RNF31 shRNA was designed with a shRNA designer tool. Two strands were annealed, followed by insertion into pcDNA6.2-GW/EmGFP-miR vector. The RNF31 cDNA containing the coding sequence was cloned by PCR, and the PCR product was cloned into the pcDNA3.1 vector. The insert was confirmed by DNA sequencing. 2.4. Colony formation assay In colony formation assay, the cells were seeded on 35-mm dishes. The cells were fixed in methanol, and then stained with crystal. Finally, positive colony formation (>50 cells/colony) was counted. 2.5. Cell migration and invasion assay Transwell migration and invasion assays were performed with 8.0-mm pore according to the manufacturers' instructions (BD Bioscience, CA). The PCa cell migration and invasion assays were performed with uncoated (migration) and coated Matrigel (invasion). The migrated and invaded PCa cells in the membrane were fixed and stained, and the cells were counted under a microscope.
2.7. RNA extraction and qRT-PCR analyses Total RNA was extracted using Trizol Reagent according to the manufacturer's protocol. The expression level of miR-503 was measured by TaqMan miRNA assays (Applied Biosystems, CA, USA) according to the provided protocol, miRNA U6 was used for normalization. RNF31 expression was measured by SYBR green qPCR assay and b-actin was used as an endogenous control. 2.8. Western blot analysis Protein concentration was measured by use of the BCA reagent kit (Merck). The protein was resolved by SDS-PAGE and transferred to a PVDF membrane, which was probed with specific primary antibody against RNF31. b-actin was blotted to show equal protein loading. 2.9. Immunohistochemistry After deparaffinization and rehydration, the tissues were washed by phosphate-buffered saline and treated with 3% H2O2 in methanol for 10 min. After being washed with distilled water, the tissues were subjected to antigen retrieval in citrate buffer and stained overnight with rabbit polyclonal anti-RNF31 antibody. The sections were incubated with goat anti-rabbit IgG for 30 min and developed with diaminobenzidine. RNF31 protein level was classified semiquantitatively combining the proportion and intensity of positively stained immunoreactive cells [21,22]. The sum of the staining intensity score and the percentage score was used to define the RNF31 protein expression levels: 0e2, low expression and 3e4, high expression [21,22]. 2.10. Prostate tumor xenograft studies We established xenografts in nude mice with the stable expressing miR-503 cells, RNF31 knockdown cells, and control cells. PCa cells were implanted into the dorsal flank of male Athymic nude mice subcutaneously. Tumor size was measured biweekly, and tumor volumes were calculated using the formula: Volume (mm3) ¼ [width2 (mm2) length (mm)]/2. Mice with tumors were killed 7 weeks after the inoculation. The xenograft tumors, and the cervical lymph nodes were collected and tumor weights were measured. DNA extraction of the cervical lymph nodes and human alu sequence PCR amplification were performed as described previously [23]. 2.11. Statistical analysis For continuous variables, Student's t-test was performed. Spearman correlation test was chosen for examining the correlations between RNF31 expression level and the clinical and pathological variables. Survival curves were carried out by the KaplaneMeier method and evaluated using the log-rank test. Identified factors were associated with survival by the Cox proportional hazard regression model. P < 0.05 was considered statistically significant. Statistical analysis was performed using SPSS 17.0 software.
2.6. Luciferase reporter assay 3. Results In brief, the miR-503-binding site in the RNF31 30 -UTR region (wild or mutant-type) was cloned downstream of the firefly luciferase gene in a pGL3-promoter vector. The luciferase assay was performed following the manufacturer's protocol. Luciferase activity was measured using the dual luciferase reporter assay system (Promega, Madison, WI).
3.1. RNF31 is a potential metastasis associated gene and is associated with PCa progression To identify whether any significant difference of RNF31 expression level exists in metastatic PCa, primary PCa, and normal
Please cite this article in press as: J. Guo, et al., miR-503 suppresses tumor cell proliferation and metastasis by directly targeting RNF31 in prostate cancer, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.127
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prostate samples, the microarray data from some available datasets were analyzed [24e28]. The data showed that RNF31 mRNA was significantly increased in primary PCa compared with normal prostate samples (Fig. 1AeC). Similarly, elevated RNF31 mRNA was found in metastatic PCa relative to primary prostate tissues (Fig. 1DeF). We further found that RNF31 was significantly with higher PCa grade, lymph node metastasis, and higher stage (Fig. 1GeI). Collectively, RNF31 is a potential metastasis associated gene in PCa. RNF31 protein expression was high in 4 of 50 patients with benign prostate hyperplasia (BPH) and 92 of 140 patients with PCa. RNF31 protein expression was overexpressed in PCa tissues compared with the BPH tissues, and the difference was statistically significant. As shown in Fig. 1J, the RNF31 staining was localized within the cytoplasm of immunoreactive prostate cells. The immunohistochemical analysis showed weak or no reactivity in normal prostate samples but strong staining in the aggressive PCa tissues. We further studied the association between RNF31 protein expression status and clinicopathological factors in PCa. The associations between RNF31 protein expression and clinicopathological factors are shown in Table S1. High expression of RNF31 protein was significantly associated with seminal vesicle invasion, the higher
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level of preoperative PSA, the positive angiolymphatic invasion, BCR, and the higher Gleason score. We further found that the high RNF31 protein expression group had a shorter overall or BCR-free survival duration compared to the low RNF31 expression group. Univariate and multivariate analysis showed that RNF31 protein expression was an independent prognostic factors for BCR-free and overall survival of patients with PCa (Tables S2 and S3). Taken together, these results indicated RNF31 overexpression is significantly associated with PCa progression and poor prognosis. 3.2. Silence of RNF31 inhibits PCa cell colony formation, migration and invasion In order to study the functional significance of RNF31 upregulation in PCa, we reduced RNF31 expression level in PCa cells and investigated its impacts on cell colony formation, migration, and invasion. We employed stable knockdown strategy targeting RNF31 in LNCaP and PC-3 cell lines. The efficiency of silence of RNF31 was investigated by western blots. We found significant decrease in colony formation in RNF31 knockdown cells compared with control cells (Fig. 2A). We also found that silence of RNF31 dramatically inhibited PC-3 and LNCaP cell migration and invasion (Fig. 2B, C). RNF31 protein was significantly decreased in the RNF31
Fig. 1. RNF31 is upregulated in PCa tissues and is correlated with PCa progression. A, B, C, D, E and F, Box plots represent RNF31 mRNA level in normal prostate, primary PCa and metastatic PCa tissues. G, H, and I, RNF31 is significantly upregulated in patients with higher PCa stage, lymph node metastasis, and higher PCa grade compared with patients with lower PCa stage, negative lymph node metastasis, and lower PCa grade respectively. J, Immunohistochemical analysis of RNF31 in benign prostate epithelia, less aggressive PCa, and aggressive PCa.
Please cite this article in press as: J. Guo, et al., miR-503 suppresses tumor cell proliferation and metastasis by directly targeting RNF31 in prostate cancer, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.127
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Fig. 2. miR-503 inhibits PCa cell proliferation, migration and invasion by targeting RNF31 in vitro. A, RNF31 knockdown can mimic the suppression of colony formation induced by miR-503 in PC-3 and LNCaP cells. B, RNF31 knockdown can mimic the suppression of migration activity induced by miR-503 in PC-3 and LNCaP cells. C, RNF31 knockdown can mimic the suppression of invasion activity induced by miR-503 in PC-3 and LNCaP cells. D, The efficiency of RNF31 knockdown and ectopic expression of miR-503 was confirmed at protein level by western blot.
knockdown cells relative to the control cells (Fig. 2D). 3.3. RNF31 as a direct target of miR-503 We used miRNA target prediction algorithms provided by miRWalk. After integrating the results, we choose miR-503 for further investigations due to its tumor suppressor properties in several malignancies [29e33]. The binding site for miR-503 at 30 UTR of RNF31 was depicted (Fig. 3A). We construct firefly luciferase reporter constructs with the 30 UTR of RNF31 mRNA, and transfected them into PCa cells with miR-503. We found that co-transfection with miR-503 can significantly reduce the luciferase activity when the construct contained the 30 UTR of RNF31 (Fig. 3B). Mutation of the binding sites can reverse the inhibitory effects. Collectively, these results indicated that RNF31 was a direct target of miR-503. We further conducted qRT-PCR and western blot in PCa cells to investigate whether ectopic expression of miR-503 can change RNF31 expression levels. RNF31 mRNA expression were dramatically inhibited in miR-503 transfectants as compared with control cells (Fig. 3C). We also found that RNF31 mRNA expression was inversely associated with the expression level of miR-503 (Fig. 3D). 3.4. miR-503 can regulate cell proliferation, invasion and migration by targeting RNF31 Our team investigate whether miR-503 can change the effects of RNF31 on PCa. We found that RNF31 upregulation can significantly abrogate the inhibition of PCa cell colony formation induced by miR-503 (Fig. 2A). Similarly, RNF31 upregulation can reverse the inhibition of PCa cell migration and invasion induced by miR-503
(Fig. 2B, C). The efficiency of miR-503 overexpression was investigated by western blots (Fig. 2D). We found that silence of RNF31 can result in similar effects induced by miR-503 overexpression in PCa cells (Fig. 2). 3.5. RNF31 plays an important role in PCa growth and metastasis in vivo To study the role of RNF31 on PCa growth in vivo, a murine xenograft model using stable RNF31 knockdown PCa cells was used. Silence of RNF31 can lead to significantly decreased tumor volume and weight relative to control group (Fig. 4AeD). Silence of RNF31 tumor tissues indicated reduced staining for RNF31 (Fig. 4E). Silence of RNF31 significantly inhibited PC-3 cell's ability to metastasize relative to control cells (Fig. 4F). Ectopic of miR-503 can result in attenuated metastasis relative to the control group, and lead to similar results induced by silence of RNF31 (Fig. 4). Our team did not find any metastases in control or experimental LNCaP xenografts. Taken together, these data suggested that RNF31 may play an important role in PCa growth and metastasis in vivo. 4. Discussion In this study, we want to identify RNF31 protein expression pattern and its association with PCa carcinogenesis, progression, and prognosis. Results indicated a statistically significant increase of RNF31 expression in PCa and metastatic PCa, indicating RNF31 plays an important role in PCa progression. To clarify the role of RNF31 in PCa, we studied the association of RNF31 staining with clinicopathological characteristics in PCa. Results indicated that RNF31 positive staining was significantly associated with PCa
Please cite this article in press as: J. Guo, et al., miR-503 suppresses tumor cell proliferation and metastasis by directly targeting RNF31 in prostate cancer, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.127
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Fig. 3. RNF31 is a direct target of miR-503 in PCa tissues. A, Computational analysis indicating that miR-503 potentially targeted RNF31. B, Relative luciferase activities were studied in PCa cells. C, Decrease in RNF31 mRNA expression by miR-503 was investigated using qRT-PCR. D, RNF31 mRNA was inversely associated with miR-503 in 140 pairs of PCa tissues using linear regression models.
Fig. 4. Ectopic expression of miR-503 inhibits tumor growth and metastasis via targeting RNF31 in vivo. A and B, Ectopic expression of miR-503 in PCa cells significantly inhibits tumor growth in a mouse xenograft model. C and D, Tumor weights of corresponding mouse xenograft models. E, RNF31 expression analysis was conducted at protein level by western blot. F, miR-503 played an important role in PCa metastasis in vivo via targeting RNF31.
Please cite this article in press as: J. Guo, et al., miR-503 suppresses tumor cell proliferation and metastasis by directly targeting RNF31 in prostate cancer, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.127
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progression. This is the first direct evidence of the association between RNF31 and clinicopathological factors of PCa. Given the significant association found between RNF31 and PCa progression, we further studied its prognostic role. Results indicated that positive staining of RNF31 was independently associated with unfavorable outcome of patients with PCa. The prognostic value of RNF31 was statistically significant in not only univariate analysis but also multivariate analysis adjusted for variables, which suggested RNF31 expression is a good biomarker to predict prognosis of PCa. These results above were the first to address the prognostic role of RNF31 in PCa. Proliferation and metastasis are two most common causes for the cancer-related death [14,34]. In order to identify the functions of RNF31, the stable RNF31 silence/overexpression PCa cell lines were constructed and the effects of them on cell proliferation, migration and invasion were studied. These results indicated that silence of RNF31 significantly inhibited the proliferation of the PCa cell lines in vitro. We found that PCa cells became less aggressive and invasive after transfected with the RNF31 knockdown construct, indicating that the RNF31 functioned as a metastasis associated gene in PCa. We also found that RNF31 knockdown can significantly inhibit tumor growth and metastasis in vivo. More and more evidence indicated miRNAs play essential roles in some processes, including differentiation, angiogenesis, proliferation, migration, and invasion. We then search the potential miRNAs which can regulate RNF31. Among of these miRNAs, miR-503 attracted our attention due to its role in several cancers. We found that miR-503 level was correlated inversely with RNF31 mRNA level. We further studied whether miR-503 can inhibit RNF31 expression level. We treated PCa cell lines with miR-503 and investigated RNF31 level. The results indicated that miR-503 can significantly negatively regulate RNF31 expression in PCa tissues. These results further indicated that the miR-503 can directly target RNF31. As expectedly, silence of RNF31 can lead to similar results induced by miR-503 overexpression in PCa. Furthermore, ectopic overexpression of RNF31 can significantly reverse the inhibitory effects of miR-503. Collectively, these results showed that miR-503 can inhibit PCa cell proliferation, migration and invasion by directly inhibiting RNF31. In conclusion, to our knowledge, this is the first study to describe the function of RNF31 in carcinogenesis, progression and prognosis of PCa. Our study also showed for the first time that miR503 may play its metastasis suppressive role through attenuation of RNF31 activity. Our investigation indicated for the first time that RNF31 could be an independent prognostic predictor to identify the poor BCR-free survival and overall survival PCa patients. Taken together, these results above suggested that RNF31 plays an important role in PCa progression and could serve as a viable therapeutic target. Acknowledgments None.
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2015.07.127. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.07.127.
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